The use of recombinant host cells in the expression of heterologous proteins has in recent years greatly simplified the production of large quantities of commercially valuable proteins, which otherwise are obtainable only by purification from their native sources.
One problem frequently encountered is the high level of proteolytic enzymes produced by a given host cell or in the culture medium. Several examples of host organisms deprived of the ability of producing specific proteolytic compounds have previously been reported. For example, International Patent Application WO 90/00192 describes filamentous fungal hosts incapable of secreting an enzymatically active aspartic proteinase, and EP 574 347 describes Aspergillus niger hosts defective in serine proteases of the subtilisin-type, pepC or pepD. Cloning and characterization of the pepC gene of Aspergillus niger has been described in Frederick et al., Gene, 125 (1993) 57-64.
So far all known prior art relating to pepC mutants and their use in production of heterologous polypeptides in filamentous fungi, particularly in Aspergillus, relate preferably to deletion mutants. This requires genetic manipulation of the host cell. It would therefore be desirable to find other means for reducing activity of specific proteases, e.g. in the form of an inhibitor. It is an object of the present invention to provide an alternative solution by providing a DNA molecule encoding an Aspergillus serine protease inhibitor, particularly a PepC inhibitor. In the yeast Saccharomyces cerevisiae an inhibitor of the proteinase yscB (pepC homologue) has been described (Schu et al., 1991, Eur. J. Biochem, 197:1-7). This inhibitor was found to be cytoplasmic. The PepC protein in Aspergillus is known as a protease produced in the vacuole. However, when Aspergillus is used as a production host the problems encountered in relation to PepC is normally that PepC can be found in the supernatant. Depending on the product produced this can be a problem for yield/stability.